Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests.
One strategy is the use of chemical insecticides with a broad range of activity. However, there are a number of disadvantages to using such chemical insecticides. Specifically, because of their broad spectrum of activity, these insecticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical insecticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances.
Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. Biopesticides comprise a bacterium which produces a toxin, a substance toxic to the pest. Biopesticides are generally less harmful to non-target organisms and the environment as a whole than chemical pesticides. The most widely used biopesticide is Bacillus thuringiensis (B.t.). B.t. is a widely distributed, rod shaped, aerobic and spore forming microorganism.
During its sporulation cycle, B.t. produces an alkali soluble protein(s) in crystal form known as a crystal delta-endotoxin(s) having a molecular weight ranging from 27-140 kd, which upon ingestion kills insect larvae. Toxic activity may reside in one or more of such crystal proteins in a given B.t. strain. Most delta-endotoxins are protoxins that are proteolytically converted into smaller toxic (truncated) polypeptides in the target insect midgut (H6fte and Whiteley, 1989, Microbiol. Rev. 53:242-255). The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on structural similarities and pesticidal specificity. The major classes are Lepidoptera-specific (cryI); Lepidoptera-and Diptera-specific (cryII); Coleoptera-specific (cryIII); Diptera-specific (cryIV) (H ofte and Whiteley, 1989, Microbiol. Rev. 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode-specific (referred to as cryV and cryVI by Feitelson et al., 1992, Bio/Technology 10:271-275).
Six cryI genes have been identified: cryIA(a), cryIA(b), cryIA(c), cryIB, cryIC, and cryID (H ofte and Whiteley, 1989, Microbiol. Rev. 53:242-255). Since cryIA(a), cryIA(b), and cryIA(c) show more than 80% amino acid identity, they are considered to be part of the cryIA group.
A number of B.t. strains have been isolated that have been found to be active against insect pests of the order Lepidoptera. B.t. subsp. kurstaki HD-1 produces bipyramidal and cuboidal crystal proteins in each cell during sporulation (L uthy et al., in Microbial and Viral Pesticides, ed. E. Kurstak, Marcel Dekker, New York, 1982, pp.35-74); the bipyramidal crystal was found to be encoded by various cryIA genes (Aronson et al., 1986, Microbiol. Rev. 50:1-50). B.t. subsp. kurstaki HD-73 contains the cryIA(c) gene for its crystal delta-endotoxin (Adang et al., 1985, Gene 36:289-300). B.t. subsp. dendrolimus HD-7 and HD-37 contain a CryIA and a CryII protein; B.t. subsp. sotto contains an alkaline soluble protein that differs from the holotype CryIA(a) protein by 24 amino acids; B.t. subsp. subtoxicus HD-10 contains CryIA and CryIB proteins; B.t. subsp. tolworthi HD-121 contains CryIA and CryII proteins; B.t. subsp. entomocidus HD-110, 4448 contains CryIA, CryIB, and CryIB proteins; and B.t. subsp. aizawai HD-68 contains CryIA proteins (H ofte and Whiteley, 1989, Microbiol. Reviews 53:242-255). Bt. subsp. aizawai HD-11 contains a Cry IA protein as well as a P.sub.2 crystal (Hofte and whiteley, 1989, Microbiol. Rev. 53:242-255). Padua, 1990, Microbiol. Lett. 66:257-262, discloses the isolation of two mutants containing two crystal delta-endotoxins, a 144 kD protein having activity against a lepidopteran pest and a 66 kD protein having activity against mosquitoes. Payne, U.S. Pat. No. 4,990,332, issued Feb. 5, 1993, discloses an isolate of B.t. PS85A1 and a mutant of the isolate, PS85A1 which both have activity against Plutella xylostella, a Lepidopteran pest and produce alkali soluble proteins having a molecular weight of 130,000 and 60,000 daltons. Payne, U.S. Pat. No. 5,045,469, issued Sep. 3, 1991 discloses a B.t. isolate designated PS81F which also produces alkali soluble proteins having a molecular weight of 130,000 and 60,000 daltons and has activity against Spodoptera exigua and T. ni; the toxin gene from PS81F appears to have little homology to the toxin gene from B.t. subsp. kurstaki HD-1. Payne, U.S. Pat. No. 5,206,166, filed Jun. 25, 1992, issued Apr. 27, 1993, discloses B.t. isolates PS81A2 and PS81RR1 which produce 133,601 and 133,367 dalton alkali-soluble proteins; both have activity against Trichoplusia ni, Spodoptera exigua and Plutella xylostella and are different from B.t subsp kurstaki HD-1 and other B.t. isolates. Payne, U.S. Pat. No. 5,169,629, filed Nov. 1, 1988, issued Dec. 2, 1992, discloses B.t. isolate PS81GG active against lepidopteran pests and which produces a bipyramidal (130,000 daltons) and a cuboidal (60,000 daltons) crystal delta-endotoxin. Payne, U.S. Pat. No. 5,188,960, filed Dec. 14, 1989, issued Feb. 23, 1993, discloses B.t. PS81I which produces a 130,000 dalton alkali soluble protein having a flagellar serotype of 7, aizawai which can be distinguished from HD-1 and is active against Spodoptera exigua, Plutella xylostella, and Choristoneura occidentalis. Bernier et al., U.S. Pat. No. 5,061,489 and WO 90/03434 discloses strain A20 producing a delta-endotoxin encoded by at least three genes: 6.6-, 5.3-, and 4.5-type genes (CryIA(a)-like, cryIA(b), and cryIA(c)). Bradfish et al., U.S. Pat. No. 5,208,017, discloses B.t. isolates PS86A1 and PS86Q3 which respectively produce alkali-soluble proteins having a molecular weight of 58,000 and 45,000 daltons and 155,000, 135,000, 98,000, 62,000, and 58,000 daltons respectively and which have activity against lepidopteran and coleopteran pests.
It is advantageous to isolate new strains of Bacillus thuringiensis so that there exists a wider spectrum of biopesticides for any given insect pest.